Myocardial infarction (MI) is a common injury that causes permanent loss of hundreds of millions of cardiomyocytes (CMs), increasing susceptibility to heart failure and sudden death. Major goals of regenerative medicine are methodologies to enhance CM recovery after MI and to restore cardiac function to heart failure patients. Heart regeneration in its natural context builds muscle by proliferation of spared CMs, facilitated by influences of supporting cell types like epicardium, endocardium, vasculature, and inflammatory cells. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received much recent attention as a target in cardiac repair strategies. Yet, until recently, virtually nothing was known about epicardial homeostasis - whether and how these cells regenerate - in any species. This deficiency has presented a barrier to understanding the epicardium and developing it for therapeutic goals. In a new study, we established the capacity of the zebrafish epicardium to vigorously regenerate, and we identified influences on this process by the cardiac outflow tract and Hedgehog signaling. While this work broke new ground in adult epicardial biology, it simultaneously revealed new challenges. First, we understand little of the details by which epicardial cells tightly connected within a contiguous sheet respond to injury and/or mitogen sources. Second, our molecular understanding of epicardial regeneration, and how epicardial cells promote muscle regeneration, is primitive. Here, we will these address central issues in epicardial biology and heart regeneration, using cutting edge screening and analysis tools in zebrafish. 1) We will apply a new ex vivo model of epicardial regeneration and a panel of new transgenic reporters that visualize epicardial cell phases in real time, to define spatiotemporal proliferation dynamics of adult epicardial cells after cardiac injury. 2) We will harness single epicardial cell transcriptome data we have generated to define new epicardial markers, initiating our work with new reagents to define the role of the epicardial marker caveolin-1 in epicardial injury responses. 3) We will identify small molecule modulators of the epicardial injury response, using an ex vivo screening strategy and new transgenic tools to visualize and manipulate the epicardium. With these approaches, we will test the hypothesis that a vigorous epicardial injury response is critical for heart regeneration. By identifying key regulators of the dynamic epicardial injury response and its impact on muscle regeneration, our work will inform approaches for comprehending and enhancing the limited regenerative response displayed by humans after MI.